Bulletin No. 13. 

U. S. DEPARTMENT OE AGRICULTURE. 

SC HTTKKATT OF ANLMAL 1XI)U8TKY. /' , -rs 

%0% 

TUBERCULOSIS INVESTIGATIONS. 



THE GROW'I'K OF TH1-: TirRERCULOSLS BACILLUS UPON ACID MEDIA. 

liy E. A. ]>;•: Schwkinitz and Mahion Douskt. 
|FUKTHEK EXPERIMENTS WlPIf AN ATTENUATED TUBERCrUOSIS BA- 
CILLUS. 

By E. A. i)K ScuwKixiT/ and E. C. Schhoedkk. 
|THE EFFECT OF TUBERCULIN INJECTIONS UPON" THE MILK OF HEALTHY 
AND DISEASED COWS. 

]>y E. A. i>K ScuwKixiTZ. 



Prepared under the direction of 

Dr. D. E. SALMON, 

Oiiel" of "tli«i Bxjii'eavi <>r ^^iiimal In<lri.-^try-. 



Isr-^nod S(^ptf-iii1)e'i- li), 1S96. 




WASHINGTON : 

GOVERNMENT PRINTING OFFICE. 
18!;G. 




Class. 
Book. 



JILS_ 



Bulletin No. 13. 



U. S. DEPARTMENT OF AGRICULTURE. 



BUREAU OF ANIMAL INDUSTRY. 



£^(^. 






TUBERCULOSIS INVESTIGATIONS. 



THK (iROWTir OF THH TURERCULOSIS BACILLUS UPON ACID MEDIA. 

By E. A. DK ScHWKiNnv. and Mauion Dokset. 
FURTIIEK. HXl'KKLMFNJ'S WITH AN ATTENUATED TUBERCULOSIS BA- 
CILLUS. 

By E. A. Die SciiWKixii/ and E. ('. SciiitoEDEH. 

THE EFFECT OF TUBERCULIN INJECTIONS UPON THE MILK OF HEALTHY 
AND DISEASED COWS. 

By E. A. 1>K SCHWKINITZ. 



Prepared iiiuk'i- the (lirection oC 
Dr. D. E. SALMON, 



Issued September 19, 1896. 




WASHINGTON : 

GOVERNMENT PRINTING OFFICE. 



18 6. 



1^ 



^^ 






net 20 »«* 



-4 



LETTER OF TRANSMITTAL. 



U. S. ])epartment of Agriculture, 

Bureau of Animal Industry, 

WaHhhujton^ D. C, July 15, ISOd. 
Sir: I hiiv^e the honor to trausmit herewith, and to recommend for 
publication as a bulletin of this Bureau, the manuscript containing a 
report of investigations upon the cultivation of the tuberculosis bacil- 
lus ui^on acid media, and of some further experiments with reference to 
the effect of tuberculin upon the milk of cows. 

These experiments have been conducted under the immediate super- 
vision of Dr. E. A. de Schweinitz, assisted by Drs. Marion Dorset and 
E. 0. Schroeder, and the publication of the results obtained will furnish 
a (convenient and desirable method of preserving and distributing the 
results of scientific investigations in this important part of the work 
of this Bureau. 

Very respectfully, 

D. E. Salmon, 

Chief of Bureau. 
Hon. J. Sterling Morton, 

/Secretary of AyricuHure. 

3 



LETTER OF SUBMITTAL. 



U. H. Department of Agriculture, 

Washington, I). C, July ], 1896. 
Sir : 1 liercwitli submit for i)ublication some results of investigations 
upon the cultivation of the tuberculosis bacillus u})on acid media, tlie 
innocuous character of an attenuated tuberculosis bacillus, and some 
further experiments Avith reference to the effect of tuberculin upon the 
milk of cows. Charts showing the average temperature reactions of 
cattle as regards breed are also appended. 
Kespectfully, 

E. A. DE SCHWEINITZ, 

Chief Biochemic Division. 
Dr. D. E. Salmon, 

Chief of Bureau of Animal Indns'ri/. 
i 



COIsTTEI^TS. 



Paga 
Tlie crowtli of the tuberculosis bacillus upou acid inedia. By E. A. do 

Scliweinitz, Pli. D., M. D., aud Mariou Dorset, M. D 7 

Introduction 7 

Observations 8 

Detn ils of experiments 9 

Further experiments with an attenuated tuberculosis bacillus. ]?y. E. A. de 

Schweinitz, Ph. D., M. D., and E. C. Schroeder, M. D. V 11 

Experiment with a monkey 11 

Results of guinea pig inoculations 12 

The effect of tuberculin injections njion the milk of healthy and diseased cows. 

By E. A. de Schweiuitz, Ph. D., M. D 15 

Analyses of nulk of cows injected with tuberculin 18 

Temperature reactions of the cows used in the milk experiments lil 



ILLUSTRATIONS. 



TLATES. 

Pago. 

PI. 1. Bacillus tuberculosis upon normal and acid media ^( 

2. Bacillus tuberculosis upon acid mettia coutaining free bydrocbloric acid, 

aud upon artificial media 9 

FIGURES. 

Fig. 1. Average temperature of 8,930 healthy and 1,191 diseased cattle injected 

with tuberculin 21 

2. Average temperature reaction of Jersey cattle 22 

3. Average temperature reaction of Holstein cattle 23 

4 . Average temperature reaction of Durham cattle 24 

5. Average temperature reaction of Ayrshire cattle 2") 

G. Average temperature reaction of Guernsey cattle 26 

7. Average temperature reaction of Grade cattle 27 

6 



TUBERCULOSIS INVESTIGATIONS. 



THE GROWTH OF THE TUBERCULOSIS BACILLUS UPON ACID MEDIA, 

By E. A. i>E SciiWKiNiTz, Ph. D., M. D., and Makion Dohset, M. D. 
INTRODUCTION. 

In the Pliiladelpliia Medical Kews of December 8, 1894, one of us 
(De Scbweiiiitz) published au article upon the attenuated bacillus tuber- 
culosis and its effect upon guinea pigs, and called attention to the fact 
that the tuberculosis germ would grow satisfactorily upon media having 
au acid rea(!tion. This ai)peared to be an important observation, and 
while we have for several years been cultivating the tuberculosis germ 
upon various media, some interesting facts have been noted which are 
Avorthy of record. 

Pawlosky, Ann. de I'lnstitut Pasteur, 1888 (and Sander) (xVrchiv fiir 
Ilyg., I>d. XVI, p. 238) have shown that vegetable broths may be used 
for the cultivation of the bacillus tuberculosis, and (in the Centralb. fiir 
Bak. und Parasit., August 8, 1895, Nos. 4, 5) Lubinski describes the 
utilization of this acid broth in the preparation of solid and liquid 
media, either with or without the addition of beef broth. 

Our cultures have been made upon the ordinary i)eptonized meat 
broth prepared from fresh meat and upon the artificial media, which, 
as one of us noted earlier, has been very useful in studying the products 
of germs. We tind that the tuberculosis bacillus will grow upon acid 

N 
beef broth which requires 21.G c. c. ^0 sodium hydrate solution to neu- 
tralize every 100 c. c. of the media. It will also grow upon acid arti- 

jST 
ficial media every 100 c. c. of which lequires 45 c. c. of ^,. sodium 

hydrate solution for neutralization. This acidity in some instances 
was due to the acids iu the meat. In the artificial media it was due to 
the acid phosphate of potassium used. 

We have also added free hydrochloric acid to the acid beef broth 

N 
and to the acid artificial media, so that from 1 c. c. to 3 c. c. ^^ hydro- 
chloric acid have been contained in every 100 c. c. of the media. The 
presence of free hydrochloric acid was tested, both before and after the 
sterilization of the media, and after the bacilli had begun to multiply, 
by means of cougo red or phloroglucin and vanillin. The congo-red 

7 



8 

test was not satisfactory in tlie beef brotli, tlie albuminoids and pos- 
sibly organic acids i)resent evidently interfering- with tlie reaction. 
The phloroglucin and vanillin test, however, proved conclusively the 
presence of free hydrochloric acid in the media, upon which there was 
a good growth of the tuberculosis bacillus. The exact quantity of the 
free hydrochloric acid present in these cultures could not be readily 
determined, as when hydrochloric acid was added to the media a por- 
tion apparently first decomposed the organic salts and phosphates. 

After the growth of the tuberculosis germ is well advanced upon 
the media described and has apparently ceased, the acid reaction of 
the cultures is still more marked, requiring for the neutralization 
of the acid substance produced by the germ to every 100 c. c. of cul- 

N 
ture 12.0 c. c. to 15 c. c. ^,. sodium hydrate, 

Lubiuski notes a decided variation in the morphology of the germ as 
grown upon the acid vegetable broth. The bacilli, he says, often 
appear in long fdaments, having, however, the same thickness that the 
germs usually possess. The filament forms described by Metschnikoff, 
Fischel, and Jones varied in thickness, showed side chains, and were 
not joined, while Lubinski reports characteristic streptococci-like for- 
mation. Jones could find this filamentous growth only after the cul- 
tures were four to five months old, Lubinski observed it in ten days. 

OBSERVATIONS. 

Our observations upon the character of the germs grown upon acid 
beef broth media and their mori^hological changes are somewhat differ- 
ent. Instead of a thread-like growth or chain growth Ave have often 
found what appeared to be spores, and sometimes the bacilli seem very 
much thicker and longer than the ordinary germs. In one culture 
examined the germ had been from generation to generation during four 
years grown on acid media and might be expected to show Lubinski's 
chain-like formation, but instead there was an apparent spore formation 
perceptible. 

The appearance of the germs grown u])on our acid media can be seen 
from the accompanying plates. PI. I, fig. 1, shows the bacillus grown 
upon glycerin beef broth for four mouths; Neisser's spore stain. PI. I, 
fig. 2, shows the bacillus grown for forty generations upon an acid 
media. 

The filament forms are not observed. There is a slight thickening 
of the bacillus and rounding at the end often noted, which would appear 
to be spore formation. There is a different change in the morphology of 
the germ as grown upon our media which confirms the conclusions of 
Lubinski that we have heteromorphic forms of the tuberculosis bacillus 
and a genuine pleomorphismus caused by the changed conditions of life. 
Bruns (Centralblatt flir Bak. und Parasitenkuude, Bd. XVII, Abth. i, 
Ko. 23) describes a germ which gives Keisser's spore reaction and in 
that particular eviilently corresponds to the ones we have pictured. 



Bui, 13, Bureau of Animal Industry. 




Plate I. 


* 






. / 

t 


<• 
1 










■\ 






Fin. 1.— Seventh generatiou in glycerine beef Inoth. 
Neisser's -spore .stain. ( x ■~'0U0.) 




' i 

1 


* 

s 

t 

*> 

• * 




• 


/ 






- 




Fig. '-i. Foitietli yreneiatiou. ai-iil media. 
Neisser's .si)i ire stain. ( ^ :200i». i 




Haines, del. 







Bacillus tuberculosis upon Neutral and Acid Media. 



Bji 13, Bureau of Animal Ind'jstry. PLATE II. 



Haines, del. 






\ 



i 



^ 



Fig. 1.— Culture foutaining free hydrochiijric acid. 
Neisser's spore stain. ( x 2000. ) 



if%. 



-^z" 



» V 



\ 

Fig. :;. — Artificial culture. Neisser's spore stain. 
( X aOOO.) 



Bacillus tuberculosis upon Acid Media containing Free Hydrochloric Acid, and 

UPON Artificial Media. 



9 

PI. TI, t\g. 1, sbows the germ grown ui)oii acid media containing free 
bydrocbloric acid. 

PJ. II, fig. 2, is a drawing of the tuberculosis germ grown on arti- 
ficial media. We do not here observe quite the same change in the 
morphology of the germ. 

DETAILS OF EXPERIMENTS. 

A more detailed description of our experiments may be interest- 
ing. The reaction of the media prepared for the cultivation of the 
tuberculosis germ was not changed in the iirocess of sterilization. A 
quantitative check of the amount of acid was made before and after 
this ])rocess. 

First experiment. — The media used for this experiment was alkaline 

and required ibr neutralization 1 c. c, <,, hydrochloric acid for every 
50 c. c. of the media. 

a. To each of 2 flasks 50 c. c. each, 5 c. c. ^7., NaOII were added. 

b. To each of 2 flasks aO c. c. each, 20 c. c. j^j, NaOH were added. 

N 

c. To each of 2 flasks 50 c. c. each, 1 c. c. ^, HCl were added. 

The six were inoculated at the same time with tuberculosis bacilli 
from (A 14). Within two weeks a good growth of the germ was noted 
over the surface of flasks a and c, while b showe<l no growth at all. 

ISecond experiment. — The media used required for neutralization 8.1 

N 
c. c. ^.v^ sodium hydrate solution for every 50 c. c. 

N 

a. To each of 2 flasks 50 c. c. were added S.4 c. c. yrv, NaOH. 

N 

b. To each of 2 flasks 50 c. c. were added 1.3 c. c. „, IlCl. 

a Hasks were neutral and b flasks acid. The two sets were inocu- 
lated from the same culture (A 15). Within two weeks the germs m 
both flasks were growing well, a perhaps a trifle better than b. At the 
end of seven weeks one flask, a, was titrated and required for neutral- 

N 
ization C.3 c. c, , ,j, NaOH. This flask had been neutral when inoculated. 

N 
b flasks required 22.5 c. c. j.., NaOH for neutralization. The increase 

N 
in acid in a flasks is represented, therefore, by G.-) c. c. |^, NaOH, and 

N 
in the b flasks by 7.5 c. c. ,q, NaOH. There was apparently a larger 

amount of the acid substance formed in the acid flasks than in those 

which were neutral at the time of inoculation. 

Third experiment. — The media used required for neutralization 0.6 

N 
c. c. jQ, NaOH for every 100 c. c. of liciuid. 



10 

N 
a. To each of 2 flasks 100 c. c, 1 c. c. j,, IICl was added. 

h. To each of 2 fla>sks 101) c. c, 2 c. c. ^^ HCl were added. 

c. To each of 2 flasks 100 c. c, .'> c. c. ,^, HCl were added. 

These flasks were all inoculated with a virulent germ, and although 

it required several weeks for the growth to begin, after four weeks 

there was growth in all of the cultures, the most acid ones seeming' to 

contain as good a growth as the others. Every 100 c. c. of the a media 

IST 
had required 11. G c. c. ,^, NaOH. 

Every 100 c. c. of the b media had required IG.C c. c. -. >, NaOH. 

N 
Every 100 c. c. of the c media had required 21.G c. c. jt^, NaOII for 

neutralization. 

Fourth experiment. — The media used in this experiment was the 

artificial media described by one of us — De Schweinitz — in 1S02, and it 

N 
required 45 c. c. ^^^ NaOH lor every 100 c. c. for neutralization. 

a 100 c. c. one flask of the media as noted. 

h 100 c. c. one flask of the media as noted with the addition of 2 c. c. 

^, hydrochloric acid. 

These were inoculated from another artificial culture, and both 
within fifteen days showed a good growth, the one containing hydro- 
chloric acid showing a slightly better growth than the other. 

Drs.Trudeau and Baldwin have pointed out that the apparent retarda- 
tion of the growth of the tuberculosis bacillus by the acid reaction of the 
media can be overcome by neutralization. This is true; but we have 
also found that if the germ is transferred from an acid culture after it 
has been cultivated on acid media for a number of months to a neutral 
nutrient fluid it will no longer grow. There would seem from this to 
be probably a poisonous principle secreted by the gei m. It may be a 
true acid or, more likely, a substance with an acid reaction. In the 
experiment, as the germs used for inoculation still floated on the 
surface of the culture they should have been alive unless some sub- 
stance especially inimical to their growth was i^roduced in the cultures. 

These experiments show clearly that the tuberculosis bacillus can be 
readily accustomed to an acid nutrient fluid, that it can easily adapt 
itself to the changed conditions of life, and that even a small amount 
of free hydrochloric acid does not interfere with its growth. They 
tend to show further, we think, that under certain conditions there 
is probably a poisonous substance produced by the germs which is 
inimical to their own life. 

The adaptability of the bacillus to a variety of media can best be 
explained by spore formation. 

March 11, 189G. 



FURTHER EXPERIMENTS WITH AN ATTENUATED TUBERCULOSIS 

BACILLUS. 

By E. A. DE ScHWKixiTZ, Ph. D., M. D., and E. C. Schroedeu, M. D. V. 

Ill the Medical News, December 8, 1894, one of us (E. A. de Scbwei- 
iiitz) briefly described an attenuated tuberculosis bacillus (human), and 
noted that while originally very virulent, by a special method of culti- 
vation it had been rendered very harmless, and not only failed to pro- 
duce disease in guinea pigs and rabbits, but in some instances imparted 
to animals inoculated with it a distinct resistance to tuberculosis upon a 
subsequent inoculation with a virulent germ. The experiments with 
this attenuated germ have been continued, and we desire to present 
further evidence of the apparent absolutely harmless character of this 
attenuated germ, reserving for a later article further reports of its 
immunizing properties. 

EXPERIMENT WITH A MONKEY. 

After a number of guinea pigs and cattle had been inoculated with 
this attenuated germ it seemed desirable to test its effect upon an ani- 
mal susceptible to tuberculosis and more like man. Accordingly a 
monkey placed at our disposal by Dr. Kinyoun was inoculated on 
September 10, 1895, with l c. c. of an emulsion of a tubercle culture 
thirtieth generation. The inoculation was made from a culture in 
which there was an active growth. 

On September 28, 1895, the injection with this material ^ c. c. of 
tuberculosis culture thirty-second generation was repeated, and again 
on January 20, 189G, 2 c. c. of tubercle culture thirty- tliird generation. 
At the seat of the first and second injections small nodules were formed, 
while no local lesion was noted at the seat of the third injection. 

On April 9 the monkey was found dead. It had not been well for 
three weeks and was quite sick during the five or six days immediately 
preceding death. It had no appetite, the hair was rough, there was 
nausea and great thirst and evidence of distress. Post-mortem exami- 
nation revealed congested lungs and an inflammatory condition of 
ileum and caecum. Dr. Schroeder says "there were no signs of tuber- 
culosis and the small nodules formed at the time of the first two 
injections had become absorbed." This attenuated germ was, there- 
fore, innocuous to the monkey, though, as noted before, it had been 

obtained from an originally very virulent germ. 

11 



12 



REST'LTS OF GTTINEA PICI INOCULATIONS. 

The guinea i)ig iiioculatioius made by one of us (Scliroeder) witli this 
attenuated germ to test its virulence were as follows: November 24, 
1S04, Nos. 280, 281, 282, and 283 received each an abdominal injection 
subcutaneously of ^ c. c. tubercle culture twenty-third generation. 

On January 25, 1895, each pig again received 1 c. c. tubercle culture 
twenty-sixth generation. October 7, 1895, guinea pig No. 281 was found 
dead. A small dry, cheesy nodule at the seat of one injection was 
noted, but there was no other evidence of tuberculosis, and death 
resulted from inflammation of the bowels. 

On January 3, 189G, guinea pig No. 282 was found dead from pneu- 
monia. There was no evidence of tuberculosis. The other two pigs, 
Nos. 280 and 283, are alive and well at this time. 

Guinea pigs Nos. 55, 5G, 57, 58, and 59 were inoculated on October 2, 
1895, subcutaneously with 1 c. c. tubercle culture thirty-second gen- 
eration. On October 17 guinea pig No. 55 was found dead, and post- 
mortem revealed a general inflammatory condition of subcutaneous 
tissue over entire abdomen. The other animals, Nos. 50, 57, 58, and 59, 
are alive and well at this writing. 

Again, January 25, 1895, guinea pigs Nos. 331 and 332 were inocu- 
lated with i c. c. tubercle culture twenty-sixth generation. On Decem- 
ber 30, 1895, guinea pig No. 331 was found dead from pneumonia. The 
liver had a peculiar mottled appearance and a few yellowish patches. 
Though a coverglass preparation showed no tubercle bacilli, two pigs, 
Nos. 121 and 122, were inoculated as checks with a portion of the liver. 
After the lapse of six months they are alive and well and show no symp- 
toms of disease. 

On February 19, 1895, guinea pig No. 332 was also found dead, death 
being due to inflammation of the bowels, and there was no evidence of 
tuberculosis. 

These inoculations made by Dr. Schroeder have confirmed the earlier 
experiments made by Dr. De Schweinitz, and agree with the following 
which have been repeated as a check on the first work. They show 
conclusively that from an originally virulent germ we have succeeded 
in obtaining an attenuated germ which, even in large doses, is appar- 
ently harmless to guinea pigs, rabbits, cattle, horses, and monkeys. 

November 15, 1895, six guinea pigs were inoculated with f c. c. each 
of an emulsion of an attenuated culture thirty-fourth generation. The 
pigs weighed as follows: 



Ouuces. 

No. 67 9 

No. 68 11 

No. 60 11 



Oiiiict's. 

No. 70 in 

No. 71 8l 

No. 81 - 12 



13 



November is No. (57 was loiiiul tlead I'rom pneumonia. The other 
animals exhibited the Ibllowiug condition on subsequent dates : 

December 13, 1S05. 



Guinea i)ig. 



Weight. 



Ko. C8 
No. fi9 
Ko. 70 
2S'o. 71 

No. 81 



Ounces. 
12 

14 
13 
10 

14 



Remark.s. 



Slight local swclliiii;; no enlarged ulands. 

Do. 

Do. 
Slight local .swelling, right inguinal gland en- 
larged. 
Slight local swelling; no enlarged glands. 



December 31, 1895. 



Guinea pig. 


Weight. 


Remarks. 


No. 68 


Oinices. 
14 
16 

15 
11 

14 


Slightly enlarged glands. 


No. 69 


No. 7(1 




No 71 


Do. 


No. 81 


Slightly enlarged gland. 





No tuberculosis was evident on autopsy of one of these, showing 
that the gerin was without pathogenic effect. The animals had steadily 
gained in weight. 

Again : 




These animals were all inoculated on February 5, 180G, with l.ic c. 
of attenuated tuberculosis culture thirty-seventh generation. On Feb- 
ruary 13, 189G, the weights of these animals were as follows : 





Guinea 


pig- 


Weight. 

Ouncen. 

9 

15i 
15 
10 
12.1 
14 
10 
14 

8 
14 


Remarks. 


No. 169 


Swelling size of a pea at point of incM illation. 
Do '^ 


No. 172 


No. 174 


Do 


No. 159 


Do. 


No. 170 


Large swelling at point of inoculation. 
Slight swelling at point of inoculation. 
Do 


No. 108 


No. 171 


No. 173 


Do 


No. 164 


Slight swelling; pneumonia. 
Do. 


No. 166 







On February 8 and 10, respectively, guinea pigs No. 167 and 176 were 
found dead from pneumonia, which they had contracted from exposure 
to cold. Subsequently guinea pigs Nos. 171, 172, 159, and 171 were 



14 

found (lead from ])neniii<)iiia. On March 10, 1890, .guinea pig- No. 100, 
wliicli had been inoculated on February 5, with the attenuated germ 
was found dead. There were no signs of tubercuk^sis, although the 
animal had been inoculated over ai month. Tlie pig died from pneu- 
monia. March 10, 1890, the record of the remaining animals was as 
follows : 



Guinea pi J 



Woifflit. 



Jleiiiarkis. 



No. 173 
No. 169 
No. 168 
No. 170 
No. 166 



Ounces. 
15 
9 
16 
10 
14 



Sliglit soro at point of iuoculatioii. 
No local lesion. 

Slight sore at i)oiut of inoculation. 
Apjtarently well. 
Do. 



Guinea pig. 


Weight. 


Guinea pig. 


Weight. 


Guinea pig. 


Weight. 


March 18. 1896: 
No 168 


Ounces. 
16 
11) 
14 
14 


]March23, 1896: 

No.170 

No. 168 


Ounces. 

m 

164 
16' 

13J 


April 7, 1896: 

No.168 

No. 166 

No.170 


Ounces. 
18 


No 170 


16 


No. 173 


No. 166 


10 


No 166 


No. 173 


No. 173 


12 









There was no enlargement of the glands indicating the beginning of 
tuberculosis, and, except a loss of weight in pigs Nos. 170 and 173, 
which was suspicious, there was no evidence of tuberculosis. 

These and other cases in which the guinea pigs were inoculated with 
the attenuated germ, and after one to two years showed no evidence of 
disease, have proved conclusively the attenuated character of this 
germ. Although this germ is so attenuated and innocuous to animals, 
its ability to grow in artificial media is as good and better than ever, 
and the tuberculin obtained from its culture is as satisfactory as that 
jjrepared from a more virulent germ. 

Cows and calves have also been inoculated with this attenuated 
germ in doses varying from 2 to 500 c. c. at a time without the produc- 
tion of tuberculosis. Nodules were freciuently formed at the i^oint of 
inoculation, which seemed, however, to be due to the mechanical 
action of the germ, and did not jnoduce any evidence of tuberculosis. 

June 29, 1890. 



THE EFFECT OF TUBERnXlN INJECTIONS UPON THE MILK OF HEALTHY 

AND DISEASED COWS. 

]\y E. A. ]>K SciiwEiNiTZ, Ph. ]>., .M. ]). 

Contiiiuiiiii^ the line of experiments given in my report. Bulletin No. 7, 
Bureau of Animal Industry, upon tlie variation in the amount of fat in 
the milk before and after the injections of tuberculin, tests were made 
on different dates upon a healthy cow, 'No. 299, with varying doses of 
tuberculin, ui)on diseased animals Nos. 145 and 161, and also upon a 
set of eight difiereut animals taken from the same milch herd — Xos. 185, 
186, 187, 189, 194, 195, 222, and 234. The latter had all been condemned 
by the tuberculin test, and preparatory to their being killed were kept 
at the station for some days, thus giving an opportunity for testing 
their milk. There was practically no variation in the fat of the milk 
from the healthy cows after the tuberculin injection. This agrees with 
our lirst experiments, and also with some tests made by Dr. Law, 
reported in Cornell University Bulletin No, 7. Neither was there any 
alteration when, as is seen from the tests in March on No. 299, large 
doses, 30 c. c. of tuberculin, were injected. The second and third injec- 
tion with tuberculin of No. 145 and 161, diseased respectively, caused 
no appreciable rise of temperature, but there was a decided decrease 
iu the amount of fat. 

In the series of January 16, 1895, however, the two animals that 
showed no rise in temperature failed to show any decrease in the milk 
fat. When the rise of temperature was noted in the others a marked 
decrease in fat was also noted. 

A comparison of the decrease in fat with the extent of the disease, 
as revealed by autopsy and given me by Dr. Smith, except in case No. 
234, a generalized one, does not apparently show anj- relationship. 
The oldest cases seemed to give the least change in fat — No. 185 — while 
the newer cases gave the largest variation. The Tables I and II show 
the quantity and composition of the various samides of milk, and the 
Tables III and lY the temperature reactions after the tuberculin injec- 
tion, for the corresponding dates. 

No. 285, an animal condemned for tuberculosis about a year ago, has 
been kept at the station since that date. At first she was injected 
with small doses of tuberculin until she ceased to give a reaction and 
was again apparently well. The injections of tuberculin were increased 
in number and (piautity, and on March 20, 1895, the date of the last 
examination of the milk, the animal received an injection of 100 c. c. 

15 



16 

Previous to that (late she liad received altotiether 505 c. c. of tuberculin. 
The hist injection caused uo change in the amount of fat or iu the 
temperature. 

The variation in fat shoukl, of course, be attributed in part to the 
fever. But that this is uot the ouly cause is also evident. The varia- 
tion is uot, judging- from the few tests made, suilicient of itself to prove 
the presence of tuberculosis, but taken iu conjunction with the rise of 
temperature might be considered as corroborative evidence. The 
tuberculin tests were made by Drs. Schroeder and Curtice, while in the 
milk analyses 1 was assisted by Mr. J. A. Emery. 

In this connection are appended several charts with curves which 
show the average variation in temperatures of a number of different 
animals. These were tabulated in February, 1895, from the reports 
received from the different States to which tuberculin had been sent. 

Chart Xo. 1 shows the average temperature of a number of animals, 
without reference to breeds. Tlie other charts are arranged so that all 
animals of the same breed are placed together. The diagnoses were 
not in all cases proved by slaughtering the animals, but in all cases 
where they were killed the autopsies confirmed the diagnoses. 

The weights of the animals were taken as the average weight, and 
the dose of tuberculin was 2 c. c. The Ilolstein cattle showed iippar 
ently a slightly higher reaction than the others. The temi)erature 
curve begins at the time of injection, and the first temperatures theu 
noted are six hours after the injection with tuberculin. 

The upper line in the charts shows the reaction of the diseased 
animals, and the lower of healthy ones. In order to make the charts 
smaller, an average was made of the temperatures of the different 
animals taken before the injection. From four to eight temperatures 
were taken of each animal, and the general average thus obtained with 
a large number of animals is given as the average normal temperature. 

In the chart giving the curve for the whole number of animals there 
is a rise of temperature noted after twenty hours. This is due to the 
Holstein chart, which is included. The late reaction noted in these 
auimals, which were from different herds, may be due to their larger size. 

Many objections have been made against the use and reliability of 
tuberculin as a diagnostic agent, the opposition coming principally 
from those who are to a great extent unfamiliar with its practical use 
or who are ouly too ready to condemn a material which, through lack 
of skill aud knowledge on their part, has perhaps given unsatisfac- 
tory results. The committee in Paris, composed of Oheveau, Leblanc, 
Mequin, Nocard, Strauss, Trasbot, aud Weber, reported as follows upon 
the principal objections to the use of tuberculin: "The use of high 
temperatures and carbolic acid in the manufacture of tuberculin makes 
it impossible that the tubeiculin, if properly prepared, should produce 
disease. It occasionally happens that tuberculin fails to give a reac- 
tion in diseased animals, but these are very exceptional cases, and occur 



17 

only sometimes when the animals are very hadly diseased and their con- 
dition conld be easily recognized, and are not of importance. Occa- 
sionally, also, apparently healthy animals show a reaction, l)ut when a 
very thorongh and careful 'autopsy is made evidence of the disease is 
usually found. In a case of an apparently healthy animal, therefore, 
one can only say in safety that the examination had not heen sufficiently 
close to discover the lesions. Again, in cases where there was appar- 
ently some other disease and the tuberculin injection caused a reaction 
a careful autopsy has shown the presence of tuberculosis, and that the 
reaction was due to the latter disease." This disposes of the objection 
that the tuberculin reaction is not characteristic. 

The statement that the tuberculin injection causes the disease to 
spread more rapidly is not warranted by facts, and in many instances 
the use of tuberculin has apparently caused an iniprovement in the 
disease. 

One animal, originally tuberculous, kept at the station of the Bureau 
of Animal Industry, has received about 3,000 c. c. tuberculin in ditfer- 
ent injections, extending over a long time. This animal is now well 
and fat and has entirely recovered from tuberculosis. 

In 1895 the International Congress for Veterinary Medicine, at Berne, 
said: "Tuberculin is a most excellent diagnostic material, and can be 
of the ntmost service in the warfare against tuberculosis." This res- 
olution was indorsed by the French Academy of Medicine and the use 
of tuberculin was generally recommended. The satisfactory reports 
received from the different States to which this Bureau has sent 
tuberculin are contirmatorj' of the results obtained, and prove that 
tuberculin is the only effective means at hand to insure a rai^id eradica- 
tion of tuberculosis in cattle. A table showing the results of the 
tuberculin injection of more than 50,000 cattle will appear in the next 
Annual Eeport of the Bureau. 

February, 1895. 

1992— ]S'o. 13 2 



18 



Table I, — Analyses of milk of cows injected ioith tuherctilin. 



Date. 


Quan- 
tity. 


Spe- 
cific 
grav- 
ity. 


Total 
solids. 


Sug- 
ar. 


Albu- 
mi- 
noids. 


Fat. 


Ash 

in 

milk. 


1894. 


c. c. 




P.ct. 


P.ct. 


P. ct. 


P.ct. 


P.ct. 


Dec. 11 


9, 655 


1.032 


13.65 


3.84 


3.14 


4.48 


0.70 


Doc. 12 


9,655 


1.031 


14.54 


4.17 


2.91 


6.31 


.70 


Dec. 13 


8,519 


1.029 


13.38 


3.84 


2.90 


5.10 


.62 


Dec. 14 


9,087 


1.029 


13.11 


4.16 


2.86 


4.10 


.66 


Dec. 18 


7, 950 


1.028 


12.74 


3.33 


2.50 


5.49 


.60 


Dec. 11 


2,000 


1.018 


21.01 


5.00 


3.2l 


15.34 


.75 


Dec. 12 


1,785 


1.025 


14.68 


2.94 


3.13 


7.73 


.75 


Dec. 13 


3,407 


1.026 


14. 95 


2. 38 


3.49 


7.16 


.72 


Dec. 14 


3,407 


1.027 


15.03 


3.12 


3.51 


7.21 


.76 


Dec. 18 


3, 407 


1.027 


16.26 


2.7t 


3.75 


8.18 


.78 


Dec. n 


450 


1.020 


18.86 


2. 50 


3.98 


12.56 


1.01 


Dec. 12 


1,025 


1.027 


12.99 


2.63 


3.05 


5.52 


.77 


Dec. 13 


1,703 


1.026 


12.77 


2.77 


2.99 


4.09 


.74 


Dec. 14 


1,703 


1.026 


12. 92 


2.94 


3.06 


4.91 


.73 


Dec. 18 


1,703 


1.026 


13.73 


2.63 


3.18 


6.35 


.81 


189.i. 
















Jan. 3 


10, 222 


1.034 


12.61 


3.57 


2.78 


4.26 


.73 


Jan 4 


9,087 


1.033 


12.34 


3.57 


2.81 


3.30 


.76 


Jan. 8 


10, 222 


1.032 


12.45 


3.57 


2.96 


3.87 


.69 


Jan. 9 


9,087 


1.034 


12.58 


3.84 


2.13 


3.86 


.67 


Jan. 10 


9,654 


1.033 


12.95 


3.84 


2.51 


4.06 


.61 


Jan. 3 


3,407 


1. 028 


16.97 


2.77 


3.36 


10.14 


.86 


Jan. 4 


3,407 


1.030 


15.40 


3.12 


3.05 


7.37 


.76 


Jan. 8 


3,691 


1.030 


15.68 


3.12 


3.56 


7.44 


.75 


Jan. 9 


3,407 


1.031 


16.74 


2.94 


3.86 


8.35 


.73 


Jan. 10 


3,123 


1.028 


15.38 


3.12 


3.58 


6.54 


.70 


Jan. 3 


1,703 


1.030 


17.54 


2.94 


3.48 


9.99 


.77 


Jan. 4 


1,992 


1.027 


13.27 


3.12 


2.97 


5.77 


.83 


Jan. 8 


2,272 


1. 029 


14.37 


2.94 


3.20 


6.53 


.68 


Jan. 9 


1,988 


1.031 


14.63 


3.33 


3.35 


6.52 


.70 


Jan. 10 


1,987 


1.031 


14.57 


3.33 


3.28 


5.91 


.71 


Feb. 18 


8,234 


1.031 


11.90 


3.33 


2.59 


3.96 


.68 


P^eb. 19 


8,519 


1.033 


12.14 


3. .57 


2.61 


3.06 


.68 


Feb. 20 


8,234 


1.032 


12.33 


3.57 


2.63 


4.07 


.68 


Mar. 4 


8,519 


1.031 


12.29 


3.57 


2.72 


3.87 


.67 


Mar. 5 


8, 945 


1.032 


11.99 


3.57 


2.68 


3.46 


.08 


Mar. 6 




1.032 


12. iO 


3 57 


2.69 


3.56 


.69 


Mar. 8 


9,087 


1.032 


11.98 


3.57 


2.61 


3.25 


.09 


Mar. 19 


4,828 


1.029 


12.84 


3.57 


3.01 


5.14 


.67 


Mar. 20 


5,112 


1.029 


14. 60 


3.57 


3.12 


6.73 


.67 


Mar. 21 


5,396 


1.029 


13.59 


3.57 


3.03 


5.81 


.66 



Total 

fat acid. 

(a) 



28.39 
30.83 
36.60 
29.21 
40.63 
38.25 
38. 81 
27.89 
27.21 
22. 99 



Volatile 
fat acid. 

(6) 



6.7 
5.0 



Acidity. 
(c) 



3.9 
5.0 
4.8 
4.8 
2.8 
5.5 
7.5 
0.1 
5.1 

3.3 



7.2 
7.2 
7.5 
1.4 
1.9 
1.9 
4.9 
5.3 
1.4 
2.0 



4.9 
5.3 
7.4 
7.7 
7.0 
7.2 
8.2 
7.4 
7.5 
5.8 
5.2 
5.1 



e.c. 
0. 132 
.146 
.108 
.112 
.132 
.100 
.136 
.130 
.134 
. 114 
.072 
.072 
.102 
.110 
.090 

.140 
.150 
.168 
.156 
. 1,54 
.100 
.156 
.122 
.138 
.138 
.130 
.132 
.124 
.130 
.124 
.128 
.132 
.150 
.154 
.156 
.160 
.156 
.106 
.096 
.098 



a Iodine absorption number. 



b Number of cubic centimeters, - Ba (oH)2, required for 1 gram of fat. 

N 
c Kuinber of cubic centimeters, — Ka oH, required for 1 cubic centimeter of n 

Table II. — Analyses of milk of cows injected with tuherculin. 



185. 


189. 


222. 


Date. 


Quan- 
tity. 


Specific 
gravity. 


Fat. 


Dat 


e. 


Quan- 
tity. 


Specific 
gravity 


Fat. 


Date. 


Quan- 
tity. 


Specific 
gravity. 


Fat. 






c. c. 










c. c. 










c. c. 






Jan. 


14 


5,111 


1.029 


5.92 


Jan. 


14 


9.655 


1.030 


4.28 


Jan. 


14 


5,111 


1.024 


10. 87 


Jan. 


15 


4,260 


1.028 


5.51 


Jan. 


15 


7,950 


1. 028 


4.29 


Jan. 


15 


5,111 


1.024 


8.20 


Jan. 


16 


4,543 


1.030 


5.50 


Jan. 


16 


7, 983 


1.030 


3.67 


Jan. 


16 


4,827 


1.030 


5.09 


Jan. 


17 


4,543 


1.030 


5.71 


Jan. 


17 


7,607 


1.028 


4.29 


Jan. 


17 


3,692 


1.029 


6.53 


Jan. 


18 


4,260 


1.029 


5.77 


Jan. 


18 


7,667 


1.027 


3.68 


Jan. 


18 


6,247 


1.029 


6.12 


186. 


194. 


229. 




14 
15 






Jan. 
Jan. 


14 
15 


1,420 
1,704 


1.028 
1.025 


10.83 
11.06 


.Tan. 
Jan. 


14 

15 


2,556 
5,679 


1.023 
1.022 


9.03 


Jan. 


375 1. 028 


5.51 


• 8.22 


Jan. 


16 


500 1. 022 


9.04 


Jan. 


16 


1,998 


1.029 


6.53 


Jan. 


16 


5,111 


1.027 


4.91 


Jan. 


17 


568 1. 022 


7.19 


Jan. 


17 


2,272 


1.030 


6.73 


Jan. 


17 


4,260 


1.027 


6.95 


Jan. 


18 


440 1. 024 


6.97 


Jan. 


18 


1,948 


L031 


5.09 


Jan. 


18 


6,816 


1.029 


4.28 


187. 


195. 


231. 


Jan. 


14 


1,998 


1.030 


8.77 


Jan. 


14 


5,111 


1.024 


6.01 


Jan. 


14 


5,111 


1.023 


8.42 


Jan. 


15 


3,123 


1.023 


12.32 


Jan. 


15 


1,704 


1.027 


7.97 


Jan. 


15 


4,543 


1. 023 


6.36 


Jan. 


16 


3,407 


1.028 


6.13 


Jan. 


16 


2,272 


1.029 


4.49 


Jan. 


16 


3,692 


1.030 


2.44 


Jan. 


17 


3,408 


1.030 


7.54 


Jan. 


17 


1,998 


1.028 


8.17 


Jan. 


17 


3,975 


1. 027 


7.36 


Jan. 


18 


3,408 


1.031 


6.16 


Jan. 


18 


2, 556 


1.030 


6.52 


Jan. 


18 


5,111 


1.027 


5.93 



19 

Table III. — Temperature reactions of the\cows used in the 7nilk experimenla. 



1894. 
Bi'forc injection. 



DiciMiibor 11 



A/fer injection. 
DiMiiiilicr 12 (iiijiitid '2 c. c. tulxTculiii at 8 ]>. in. DfciiiilKT 11) 



18U5. 
lief ore injection. 



JainiMiv 



After injection. 
January 8 (in.jci-tt'd "2 c. <•. Uibrrculin at 8 i>. in. January 7) 



Before injection. 



February 18. 



After injection. 
February 19 (iu.jected 2 c. c. tuborculiu at 9 p. in. February 18) . 



liefore injection. 



aiar.b i. 



After injection. 
!Marcli f) (iujectt'd ;!() c. c. tuberculin March 4) . 



1.00 p.m. 
3.00 p.m. 
5.00 p.m. 



8.00 a.m. 
10.00 a.m. 
12.00 m. 

2.00 p.m. 

4.00 p.m. 



1.00 p.m. 
;).00p. m. 
5.00 p.m. 



8.00 a.m. 
10.00 a. m. 
12.00 m. 

2.00 p.m. 

4.00 p.m. 

9.00 a.m. 
12.00 m. 
4.00 p.m. 
9.00 p.m. 



8.00 a.m. 

9.00 a.m. 
10.00 a.m. 
11.00 a.m. 
12.00 m. 

1.00 p.m. 

2.00 p.m. 

3.00 p.m. 

4.00 p.m. 

5.00 p.m. 

8.30 a. m. 
12.00 m. 
4.30 p. m. 



8.00 a. m. 

9.00 a. ra. 
10.00 a. m. 
12.00 m. 

2.00 p.m. 

4.00 p.m. 

5.00 p. m. 



No.299. No. 161. No. 145 



102.3 
102. 
101.5 



101.0 
100.8 
101.2 
101.0 
101.4 



101.0 
101.0 
101. 2 



101.5 
101.0 
100.4 
101.4 
101.3 

101.6 
100.6 
101. 9 
101.2 



101.6 
102.7 
102. 6 
100. 2 
100.2 
101.2 
101.5 
101.8 
101.8 
101.4 

101.2 
100.2 
101.2 



101.5 
102.0 
99.2 
100. 2 
101.2 
101.5 
101.9 



101.3 
98.2 
99.4 



101.0 
101.2 
100.6 
102.0 
101.0 



102.4 
102.6 
102.7 



1(11.8 
101.7 

100. 8 

101. 
101.5 



101.6 
97.3 
98.5 



101.4 
101.0 
102. 5 
101.2 
101.6 



100.6 
101.2 
101.4 



105.2 
105. 
102. 6 
101.5 
101.1 



20 



'J'aiu.k Iv^ — TemperatHrc reactions of the cows used in the milk exjurlmenls. 
[First test of tubercalous cows.] 




[Secontl test of tuberculous cows.] 



1895. 






















Before injection . 






















January 15 ..•••■.. 


4.n0]). in. 
9.00 p.m. 


102.2 
102. 


102. 5 
101.8 


101.8 
100. 8 


102. 8 
102.5 


102.0 
101.8 


101.4 
101.8 


101.4 
101.0 


101.6 
101.0 


100.2 




101.4 


After injection. 






















January 16 (iu.jectcd 2 






















c. c. tuberculin 11 p. m. 






















January 15) 


8.00 a.m. 


101.5 


102.0 


99. C 


102.0 


102. G 


105.2 


104.7 


103.2 


105.3 




8.30 a. ni. 


101.8 


103.2 


101.0 


102.0 


101.8 


105.2 


105.2 


104.0 


105.4 




9.00 a. m. 


102. 4 


103.2 


101.2 


103.3 


102.6 


105.2 


105.2 


104.6 


105. 4 




9.30 a.m. 


102. 2 


103.5 


101.2 


103.2 


102. 8 


105.3 


105. 4 


105.4 


105.8 




10.00 a. 7n. 


]02.2 


103.6 


101.2 


103.4 


102.8 


105.4 


105. 3 


105. 2 


106.0 




10.30 a.m. 


102.2 


103.0 


101.6 


104.2 


103.0 


105. 2 


106.0 


105.5 


100. 




11.00 a.m. 


102.4 


103.2 


101.5 


104.6 


104.2 


105.1 


106.0 


105. G 


106. 2 




11.30 a.m. 


102. 6 


102.9 


101.2 


104.2 


104.4 


105.0 


104.6 


105.4 


106.4 




12.00 m. 


103. 


102.8 


103. 2 


104.2 


104.4 


104.8 


105. 3 


105.0 


106.3 




12.30 p.m. 


103.6 


102.6 


103.6 


104.6 


104.3 


104.6 


10.3. 5 


104.8 


106.2 




1.30 p.m. 


103.5 


103.0 


103.9 


104. 8 


104.6 


104.5 


105. 5 


105.2 


lOG.O 




2.00 p.m. 


103.4 


102.0 


104.5 


104.8 


104.6 


104.0 


104. 6 


104.6 


105.6 




2.30 p.m. 


103.6 


101.6 


104.6 


104.4 


104.2 


104.0 


103. 4 


103.5 


105.2 




3.00 p.m. 


102.4 


100.0 


105.0 


100. 


102.8 


103.4 


103.5 


102.4 


104.6 




3.30 p.m. 


101.9 


99.5 


105.0 


99.6 


102.4 


103.0 


103.2 


102.5 


104.2 




4.00 p.m. 


101.6 


99.3 


104.6 


99.4 


102.0 


103.2 


103. 2 


102.6 


1C4.0 




4.30 p.m. 


102.8 


99.6 


104.5 


99.4 


102. 


103.0 


103.2 


102.6 


104.2 



21 





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FAHR. 
107° 

106° 

.5 
105° 

.5 
104° 

.5 
103° 

.5 
102° 

101° 

.5 

100° 



23 



FAHh 
107 

106 

105 

104 

103 

102 

101 

100 




oOi °Ca, °o, ''O, oO, °o, o, or«j 








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